The long term goal of this Project is a mechanistic understanding is a dynamic three dimensional (3D) model of each NT molecule and its complexes with ligands. We plan to proceed with the following coordinated steps: 1. The cental aim is to develop 3D molecular models that incorporate data dn principles from structure-function probing of membrane proteins in general, and NT-s in particular. A consensus 3D model structure of NT-s will be developed, as well as specific models for DAT, SET, NET, GAT-1. Models obtained at each stage, informed by the results from experimental probing (and especially from our collaborative work within the PPG), will serve as working hypotheses for the design of further explorations (e.g., by SCAM, Zn-binding PPG), will serve as working hypotheses for the design of further explorations (e.g., by SCAM, Zn-binding site engineering and other mutation methods). In turn, the experimental data will inform refinements of both structure and function models. Stages of the modeling include: i) a 2/o structure map to guide experimental explorations of transmembrane (TM) segment topology and identification of local structure of TM segments and of their connecting "loop" regions; ii) A map of protein-lipid and protein-protein interfaces to guide experimental explorations of the bundling of TM segments into TM domains of the NT-s; iii) identification of local adjacencies of TM segments from experiments and from computations of correlated and evolutionarily revertant mutations; iv) Exploration of inter-segment interactions and criteria for bundling. 2. We will identify in the structures of NT-s and their ligands, reactivity properties that determine the modes of NT-ligand interactions. These studies of molecular determinants for ligand activity will connect the NT modeling with results from experimental structure-function probing of NT-s in all the other Projects of this PPG. A practical goal is to develop design principles for novel ligands with discriminant pharmacological actions (e.g., cocaine antagonists). 3. To achieve (ultimately) an integration of the structural models with the mechanistic details of substrate, inhibition and electrophysiological modulation obtained in this PPG, increasingly larger portions of the 3D molecular models that are constructed iteratively in Specific Aim #1, will be explored computationally to identify local and global reactivity properties (pertaining to ligand-dependent mechanisms), and to characterize comparative changes in their dynamic properties.